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- EMDB-4611: Cryo-EM structure of calcium-bound mTMEM16F lipid scramblase in d... -

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Basic information

Entry
Database: EMDB / ID: EMD-4611
TitleCryo-EM structure of calcium-bound mTMEM16F lipid scramblase in digitonin
Map data
Sample
  • Complex: mTMEM16F
    • Protein or peptide: Anoctamin-6Calcium-dependent chloride channel
  • Ligand: CALCIUM IONCalcium
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Function / homology
Function and homology information


calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / activation of blood coagulation via clotting cascade ...calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / activation of blood coagulation via clotting cascade / negative regulation of cell volume / intracellularly calcium-gated chloride channel activity / bone mineralization involved in bone maturation / pore complex assembly / cholinergic synapse / plasma membrane phospholipid scrambling / voltage-gated monoatomic ion channel activity / bleb assembly / positive regulation of phagocytosis, engulfment / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity / positive regulation of endothelial cell apoptotic process / positive regulation of monocyte chemotaxis / dendritic cell chemotaxis / chloride transport / phospholipid translocation / chloride channel activity / chloride channel complex / monoatomic cation transport / sodium ion transmembrane transport / regulation of postsynaptic membrane potential / positive regulation of bone mineralization / chloride transmembrane transport / Neutrophil degranulation / synaptic membrane / establishment of localization in cell / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / identical protein binding / metal ion binding / plasma membrane / cytosol
Similarity search - Function
Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAlvadia C / Lim NK / Clerico Mosina V / Oostergetel GT / Dutzler R / Paulino C
Funding support Switzerland, Netherlands, 2 items
OrganizationGrant numberCountry
European Research Council339116, AnoBest. Switzerland
Netherlands Organisation for Scientific Research740.018.016 Netherlands
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.
Authors: Carolina Alvadia / Novandy K Lim / Vanessa Clerico Mosina / Gert T Oostergetel / Raimund Dutzler / Cristina Paulino /
Abstract: The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium- ...The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca define the ligand-free closed conformation of the protein and the structure of a Ca-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.
History
DepositionFeb 13, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6qp6
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4611.png

Downloads & links

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Map

FileDownload / File: emd_4611.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.012 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.05
Minimum - Maximum-0.2823333 - 0.3519569
Average (Standard dev.)0.00004834267 (±0.010796614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 222.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z222.640222.640222.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2820.3520.000

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Supplemental data

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Mask #1

Fileemd_4611_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 used during refinement and for...

Fileemd_4611_half_map_1.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation mTMEM16F_dig_Ca.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 used during refinement and for...

Fileemd_4611_half_map_2.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation mTMEM16F_dig_Ca.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mTMEM16F

EntireName: mTMEM16F
Components
  • Complex: mTMEM16F
    • Protein or peptide: Anoctamin-6Calcium-dependent chloride channel
  • Ligand: CALCIUM IONCalcium
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: mTMEM16F

SupramoleculeName: mTMEM16F / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T cells
Molecular weightTheoretical: 212 KDa

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Macromolecule #1: Anoctamin-6

MacromoleculeName: Anoctamin-6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 106.367727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MQMMTRKVLL NMELEEDDDE DGDIVLENFD QTIVCPTFGS LENQQDFRTP EFEEFNGKPD SLFFTDGQRR IDFILVYEDE SKKENNKKG TNEKQKRKRQ AYESNLICHG LQLEATRSVS DDKLVFVKVH APWEVLCTYA EIMHIKLPLK PNDLKTRSPF G NLNWFTKV ...String:
MQMMTRKVLL NMELEEDDDE DGDIVLENFD QTIVCPTFGS LENQQDFRTP EFEEFNGKPD SLFFTDGQRR IDFILVYEDE SKKENNKKG TNEKQKRKRQ AYESNLICHG LQLEATRSVS DDKLVFVKVH APWEVLCTYA EIMHIKLPLK PNDLKTRSPF G NLNWFTKV LRVNESVIKP EQEFFTAPFE KSRMNDFYIL DRDSFFNPAT RSRIVYFILS RVKYQVMNNV NKFGINRLVS SG IYKAAFP LHDCRFNYES EDISCPSERY LLYREWAHPR SIYKKQPLDL IRKYYGEKIG IYFAWLGYYT QMLLLAAVVG VAC FLYGYL DQDNCTWSKE VCDPDIGGQI LMCPQCDRLC PFWRLNITCE SSKKLCIFDS FGTLIFAVFM GVWVTLFLEF WKRR QAELE YEWDTVELQQ EEQARPEYEA QCNHVVINEI TQEEERIPFT TCGKCIRVTL CASAVFFWIL LIIASVIGII VYRLS VFIV FSTTLPKNPN GTDPIQKYLT PQMATSITAS IISFIIIMIL NTIYEKVAIM ITNFELPRTQ TDYENSLTMK MFLFQF VNY YSSCFYIAFF KGKFVGYPGD PVYLLGKYRS EECDPGGCLL ELTTQLTIIM GGKAIWNNIQ EVLLPWVMNL IGRYKRV SG SEKITPRWEQ DYHLQPMGKL GLFYEYLEMI IQFGFVTLFV ASFPLAPLLA LVNNILEIRV DAWKLTTQFR RMVPEKAQ D IGAWQPIMQG IAILAVVTNA MIIAFTSDMI PRLVYYWSFS IPPYGDHTYY TMDGYINNTL SVFNITDFKN TDKENPYIG LGNYTLCRYR DFRNPPGHPQ EYKHNIYYWH VIAAKLAFII VMEHIIYSVK FFISYAIPDV SKITKSKIKR EKYLTQKLLH ESHLKDLTK NMGIIAERIG GTVDNSVRPK LE

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 2 / Formula: P1O
Molecular weightTheoretical: 566.728 Da
Chemical component information

ChemComp-P1O:
1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DDPC, phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.3 mg/mL
BufferpH: 7.5
Details: 0.1% digitonin, 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA. 1 mM CaCl2 were added before freezing.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: at 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 49407
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 105.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 7 / Number real images: 5633 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1348247
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5yob

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 9 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 219302
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5yob

RefinementSpace: REAL
Output model

PDB-6qp6:
Cryo-EM structure of calcium-bound mTMEM16F lipid scramblase in digitonin

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